Herpesviruses continue to be a major agent of debilitating human diseases, in both healthy and immunocompromised individuals. The development of new anti-viral agents to block infection or propagation is held back by inadequate knowledge of key intracellular structures and steps in Herpes assembly and infection. Our laboratory has pioneered the combination of high resolution cryo-electron microscopy (cryo-EM) of viruses with cryo-electron tomography (cryo-ET) of intracellular capsid intermediates. These advances will be applied to the Herpes simplex virus type 1 (HSV-1) along with a similar and simpler dsDNA virus, bacteriophage P22 of Salmonella as a testbed for methodology development and testing. Although both viruses have substantially different genome and physical sizes, HSV-1 and P22 share similar capsid assembly and infection pathways. In HSV-1, a layer of tegument proteins surrounds the capsid together with the external membrane layer needed for initial cell fusion. However, both capsids consist of a portal inserted at one vertex of their capsid shell, which is assembled with the assistance of internal scaffolding proteins. The scaffolding proteins are transient components and exit the particle while DNA is being packaged. Packaging takes place through the portal, which also serves as the exit route for DNA ejection from the capsid into the cell. Our goal is to determine the structure and functional role for each of the capsid proteins involved in capsid assembly and DNA injection, including the non-icosahedral components whose structures have not been accessible. We put particular emphasis on capsid structures functioning in DNA packaging and ejection. In this proposal, we will a) elucidate the mechanisms governing initiation of a portal-containing capsid shell, shell growth, and conformational maturation, by studying the interactions among shell, scaffolding, portal, and pilot (if present) proteins;and b) to obtain structural snapshots of virus intermediates during infection and assembly in their native cell environments. These experiments employ state-of-the-art cryo-electron microscopes for data collection and advanced image processing techniques to obtain the highest possible resolution structures. Our technical approaches and biological insights will be transformative in the study of viruses, not only in biochemically purified states, but also in their native cellular environments.